Tinton S, Calderon P B
Unité de Pharmacocinétique, Métabolisme, Nutrition et Toxicologie, Université Catholique de Louvain, Brussels, Belgium.
Int J Toxicol. 2001 Jan-Feb;20(1):21-7. doi: 10.1080/109158101750103350.
Hypoxia causes a rapid and reversible inhibition of translation in freshly isolated rat hepatocytes. This inhibition is neither due to an ATP loss nor to an increase in cell death. Because protein synthesis is mainly regulated by reversible phosphorylation of initiation and/or elongation factors, we investigated whether translation inhibition by hypoxia may be related to changes in the phosphorylation status of proteins. Whatever the incubation conditions, three phosphoreactive bands (molecular weights 220, 129, and 83 kDa) were detected by antiphosphotyrosine antibodies. The phosphorylation in the 129- and 83-kDa bands, however, was significantly and progressively decreased under hypoxia. Although this time-dependent decrease was sensitive to changes in oxygen tension, it occurred after the early protein synthesis inhibition caused by hypoxia. Moreover, sodium orthovanadate prevented tyrosine dephosphorylation in hypoxic cells, but did not restore the depressed protein synthesis caused by hypoxia. Under aerobic conditions, orthovanadate inhibited the synthesis of proteins, confirming that protein phosphorylation is a major mechanism involved in translational regulation. Once again, this inhibitory effect occurred only after 90 minutes of incubation whereas hypoxia inhibits the protein synthesis at the beginning of the incubation. Labeling cells with [33-32P]-ortho-phosphoric acid allowed detection of several phosphorylated proteins that appeared under hypoxia. Because they were not recognized by the phosphotyrosine antibodies, we suggest that serine/threonine residues of key proteins may be the putative hypoxic targets.
缺氧会导致新鲜分离的大鼠肝细胞中蛋白质合成迅速且可逆地受到抑制。这种抑制既不是由于ATP的损失，也不是由于细胞死亡增加所致。由于蛋白质合成主要受起始因子和/或延伸因子可逆磷酸化的调节，我们研究了缺氧引起的翻译抑制是否可能与蛋白质磷酸化状态的变化有关。无论培养条件如何，抗磷酸酪氨酸抗体均能检测到三条磷反应带（分子量分别为220、129和83 kDa）。然而，在缺氧条件下，129 kDa和83 kDa条带中的磷酸化显著且逐渐降低。尽管这种随时间的降低对氧张力的变化敏感，但它发生在缺氧引起的早期蛋白质合成抑制之后。此外，原钒酸钠可防止缺氧细胞中的酪氨酸去磷酸化，但不能恢复缺氧引起的蛋白质合成抑制。在有氧条件下，原钒酸钠抑制蛋白质合成，证实蛋白质磷酸化是参与翻译调控的主要机制。同样，这种抑制作用仅在孵育90分钟后才出现，而缺氧在孵育开始时就抑制蛋白质合成。用[33 - 32P] - 正磷酸标记细胞，可检测到缺氧条件下出现的几种磷酸化蛋白质。由于它们不被磷酸酪氨酸抗体识别，我们认为关键蛋白质的丝氨酸/苏氨酸残基可能是假定的缺氧靶点。
